Tuning Physical Properties of GelMA Hydrogels through Microarchitecture for Engineering Osteoid Tissue.

Gelatin methacryloyl (GelMA) hydrogels have gained significant attention due to their biocompatibility and tunable properties. Here, a new approach to engineer GelMA-based matrices to mimic the osteoid matrix is provided. Two cross-linking methods were employed to mimic the tissue stiffness: standard cross-linking (SC) based on visible light exposure (VL) and dual cross-linking (DC) involving physical gelation, followed by VL. It was demonstrated that by reducing the GelMA concentration from 10% (G10) to 5% (G5), the dual-cross-linked G5 achieved a compressive modulus of ∼17 kPa and showed the ability to support bone formation, as evidenced by alkaline phosphatase detection over 3 weeks of incubation in osteogenic medium. Moreover, incorporating poly(ethylene) oxide (PEO) into the G5 and G10 samples was found to hinder the fabrication of highly porous hydrogels, leading to compromised cell survival and reduced osteogenic differentiation, as a consequence of incomplete PEO removal.

Experimental and numerical study on failure mechanisms of bone simulants subjected to projectile impact.

Analyses of the human bones failure mechanisms under projectile impact conditions can be made through performing of a large number of ballistic trials. But the amount of data that can be collected during ballistic experiments is limited due to the high dynamics of the process and its destructive character. Numerical analyses may support experimental methodologies allowing to better understand the principles of the phenomenon. Therefore, the main aim of the study was to create and to verify a numerical model of commercially available synthetic bone material-Synbone®. The model could be used in the future as a supporting tool facilitating forensic studies or designing processes of personal protection systems (helmets, bulletproof vests, etc.). Although Synbone® is commonly used in the ballistic experiments, the literature lacks reliable numerical models of this material. In order to define a numerical model of Synbone®, mechanical experiments characterizing the response of the material to the applied loads in a wide range of strains and strain rates were carried out. Based on the mechanical tests results, an appropriate material model was selected for the Synbone® composite and the values of constants in its equations were determined. Material characterization experiments were subsequently reproduced with numerical simulations and a high correlation of the results was obtained. The final validation of the material model was based on the comparison of the ballistic impact experiments and simulation results. High similarity obtained (relative error lower than 10%) demonstrates that the numerical model of Synbone® material was properly defined.

Modified Histopathological Protocol for Poly-ɛ-Caprolactone Scaffolds Preserving Their Trabecular, Honeycomb-like Structure.

Poly-ɛ-caprolactone (PCL) is now widely studied in relation to the engineering of bone, cartilage, tendons, and other tissues. Standard histological protocols can destroy the carefully created trabecular and honeycomb-like architecture of PCL scaffolds, and could lead to scaffold fibers swelling, resulting in the displacement or compression of tissues inside the scaffold. The aim of this study was to modify a standard histopathological protocol for PCL scaffold preparation and evaluate it on porous cylindrical PCL scaffolds in a rat model. In 16 inbred Wag rats, 2 PCL scaffolds were implanted subcutaneously to both inguinal areas. Two months after implantation, harvested scaffolds were first subjected to µCT imaging, and then to histopathological analysis with standard (left inguinal area) and modified histopathological protocols (right inguinal area). To standardize the results, soft tissue percentages (STPs) were calculated on scaffold cross-sections obtained from both histopathological protocols and compared with corresponding µCT cross-sections. The modified protocol enabled the assessment of almost 10× more soft tissues on the scaffold cross-section than the standard procedure. Moreover, STP was only 1.5% lower than in the corresponding µCT cross-sections assessed before the histopathological procedure. The presented modification of the histopathological protocol is cheap, reproducible, and allows for a comprehensive evaluation of PCL scaffolds while maintaining their trabecular, honeycomb-like structure on cross-sections.

Preparation of a Ceramic Matrix Composite Made of Hydroxyapatite Nanoparticles and Polylactic Acid by Consolidation of Composite Granules.

Composites made of a biodegradable polymer, e.g., polylactic acid (PLA) and hydroxyapatite nanoparticles (HAP NPs) are promising orthopedic materials. There is a particular need for biodegradable hybrid nanocomposites with strong mechanical properties. However, obtaining such composites is challenging, since nanoparticles tend to agglomerate, and it is difficult to achieve good bonding between the hydrophilic ceramic and the hydrophobic polymer. This paper describes a two-step technology for obtaining a ceramic matrix composite. The first step is the preparation of composite granules. The granules are obtained by infiltration of porous granules of HAP NPs with PLA through high-pressure infiltration. The homogeneous ceramic-polymer granules are 80 µm in diameter, and the composite granules are 80 wt% HAP NPs. The second step is consolidation of the granules using high pressure. This is performed in three variants: Uniaxial pressing with the pressure of up to 1000 MPa at room temperature, warm isostatic compaction (75 MPa at 155 °C), and a combination of the two methods. The combined methods result in the highest densification (99%) and strongest mechanical properties; the compressive strength is 374 MPa. The structure of the ceramic matrix composite is homogeneous. Good adhesion between the inorganic and the organic component is observable using scanning electron microscopy.

Impact of the Balloon Inflation Time and Pattern on the Coronary Stent Expansion.

OBJECTIVES: To assess the expansion pattern of coronary stents by using different balloon inflation times and pressures. BACKGROUND: The selection of coronary stent size and its proper deployment is crucial in coronary artery interventions, having an impact on the success of the procedure and further therapy. METHODS: Ten pairs of different stents were deployed under nominal pressure using sequential (5, 5, 10, and 10 seconds of repeated inflations, thus 30 seconds of summarized time) and continuous (30 seconds) deployment pattern. After each given time-point, intraluminal stent measurements were performed by optical coherence tomography (OCT) and intravascular ultrasound (IVUS). RESULTS: Both in-stent diameters and cross-section areas (CSA) of paired stents measured by OCT at all sequential time-points were significantly smaller compared to given manufacturers charts' values (90% to 94% for diameters and 81% to 88% for CSA, p<0.05). Significant increase of in-stent diameter and CSA was observed across the step-by-step deployment pattern. In-stent lumen measurements were significantly larger when sequential deployment pattern was applied compared to continuous deployment. Additional measurements were also done for overlapping segments of stents, showing smaller in-stent measurements of the latter compared to nonoverlapping segments. Validation of OCT and IVUS measurements using a phantom metallic tube showed perfect reproducibility with OCT and overestimation with IVUS (8% for diameters and 16% for CSA). CONCLUSIONS: Stent diameter after deployment is time-dependent and not only pressure-dependent. Different stent expansion behavior, depending on the applied deployment pattern (sequential and nonsequential), was observed.

3D-Printing of Functionally Graded Porous Materials Using On-Demand Reconfigurable Microfluidics.

Tailoring the morphology of macroporous structures remains one of the biggest challenges in material synthesis. Herein, we present an innovative approach for the fabrication of custom macroporous materials in which pore size varies throughout the structure by up to an order of magnitude. We employed a valve-based flow-focusing junction (vFF) in which the size of the orifice can be adjusted in real-time (within tens of milliseconds) to generate foams with on-line controlled bubble size. We used the junction to fabricate layered and smoothly graded porous structures with pore size varying in the range of 80-800 µm. Additionally, we mounted the vFF on top of an extrusion printer and 3D-printed constructs characterized by a predefined 3D geometry and a controlled, spatially varying internal porous architecture, such as a model of a bone. The presented technology opens new possibilities in macroporous material synthesis with potential applications ranging from tissue engineering to aerospace industry and construction.

In vivo and in vitro study of a novel nanohydroxyapatite sonocoated scaffolds for enhanced bone regeneration.

There still remains a need for new methods of healing large bone defects, i.e., gaps in bone tissue that are too big to naturally heal. Bone regrowth scaffolds can fill the bone gap and enhance the bone regeneration by providing cells with a support to for new tissue formation. Coating of the scaffolds surface with nanocrystalline hydroxyapatite may enhance the osteoinductivity or osteoconductivity of such scaffolds. Here we present the sonocoating method to coat scaffolds with bioactive hydroxyapatite nanoparticles. We show a method, where the material to be coated is immersed in a colloidal suspension of nanoparticles with mean sizes of 10 nm and 43 nm in water, and high-power ultrasound waves are applied to the suspension for 15 min at 30 °C. High power ultrasounds lead to growth of cavitation bubbles in liquid, which implode at a critical size. The implosion energy propels the nanoparticles towards the material surface, causing their attachment to the scaffold. Using this technique, we produced a uniform layer of nanohydroxyapatite particles of thickness in the range 200 to 300 nm on two types of scaffolds: a porous ß-TCP ceramic scaffold and a 3D-printed scaffold made of PCL fibers. In vivo tests in rabbits confirmed that the novel coating strongly stimulated new bone tissue formation, with new bone tissue occupying 33% for the nHAP-coated PCL scaffold and 68% for the nHAP-coated ß-TCP after a 3-month test. The sonocoating method leads to formation of a bioactive layer on the scaffolds at temperature close to room temperature, very short time and in water. It is a green technological process, promising for bone tissue regeneration applications.

Formation of calcium phosphate coatings within polycaprolactone scaffolds by simple, alkaline phosphatase based method.

The paper presents a novel approach to the production of calcium phosphate coatings of scaffolds. Mineral deposits were formed during incubation of polycaprolactone (PCL) scaffolds with bovine intestinal alkaline phosphatase in sodium glycerophosphate and calcium chloride medium. To modify hydrophobic surface of scaffolds and intensify attachment of coating, scaffolds were incubated at 50 °C (thermal activation, TA) or at 37 °C after short exposition to lipase (lipase activation, LA). Micro-computed tomography observations demonstrated that both methods resulted in deposition of mineral on the surface of external and internal walls of the scaffolds. Precipitate formed after thermal and lipase activation contained particles with average size of 200-400 nm, and the shape of donuts. In thermal activated PCL coatings X-ray diffraction disclosed peaks typical for hydroxyapatite (HAp), while after lipase activation these peaks could be precisely defined only if left for 6 days in the incubation medium. The Fourier-transform infrared spectroscopy suggested crystalline structure of HAp both after thermal and lipase activation. The adherence of bone marrow mesenchymal stem cells was initially higher on coated than pristine PCL, but during 7 days of culture the cell number increased and was similar on all tested samples. Alkaline phosphatase activity, considered as a sign of osteogenic differentiation, measured on PCL samples after 7 days was 2-3 times lower on pristine PCL than on the coated samples, but after 2 weeks increased significantly and reached similar value as on the calcium phosphate substrates.

X-ray physics-based CT-to-composition conversion applied to a tissue engineering scaffold, enabling multiscale simulation of its elastic behavior.

Nowadays, the assessment of the mechanical competence of tissue engineering scaffolds based on computer simulations is a well-accepted technology. Typically, such simulations are performed by means of the Finite Element (FE) method, with the underlying structural model being created based on micro-computed tomography (microCT). Here, this analysis modality is applied to a new, ternary composite, consisting of PHBV, i.e. poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PLGA, i.e. poly(lactic-co-glycolide), as well as of TCP, i.e. tricalcium phosphate hydrate. The studied scaffold structure is made up by fibers of this new composite material, manufactured by means of the rapid prototyping method. The data collected from microCT is utilized for adequately defining the mechanical properties of the FE model. In particular, the three-dimensional field of grey values is interpreted in terms of the underlying field of attenuation coefficients, taking into account the photon energy employed in microCT imaging, eventually allowing for calculation of the three-dimensionally distributed, voxel-specific composition of the studied material. For the sake of keeping the FE simulations as efficient as possible, groups of voxels are combined into one finite element; the grey value of the latter is obtained by volume averaging. Employing a two-step micromechanical homogenization scheme, the experimentally accessible stiffness of the three constituents (PHBV, PLGA, and TCP) is then, finite element by finite element, upscaled to the composition-dependent stiffness of the composite material. The plausibility and adequacy of the FE model is demonstrated by simulating the effects of uniaxial compression on the scaffold structure, in terms of resulting stress and strain fields, highlighting the importance of the fiber junctions (as they are the mechanically most stressed regions), and that neglecting the material heterogeneity would lead to a potentially significant underestimation of stresses and strains. Finally, a comparison is made of the employed analysis modality of microCT data with a previously pursued, simplified analysis strategy, highlighting the conceptual superiority of the former, and pointing out the application limits of the latter.

The influence of chemical polishing of titanium scaffolds on their mechanical strength and in-vitro cell response.

Selective Laser Melting (SLM) is a powder-bed-based additive manufacturing method, using a laser beam, which can be used to produce metallic scaffolds for bone regeneration. However, this process also has a few disadvantages. One of its drawbacks is the necessity of post-processing in order to improve the surface finish. Another drawback lies in the removal of unmelted powder particles from the build. In this study, the influence of chemical polishing of SLM fabricated titanium scaffolds on their mechanical strength and in vitro cellular response was investigated. Scaffolds with bimodal pore size (200 µm core and 500 µm shell) were fabricated by SLM from commercially pure titanium powder and then chemically treated in HF/HNO3 solutions to remove unmelted powder particles. The cell viability and mechanical strength were compared between as-made and chemically-treated scaffolds. The chemical treatment was successful in the removal of unmelted powder particles from the titanium scaffold. The Young's modulus of the fabricated cellular structures was of 42.7 and 13.3 GPa for as-made and chemically-treated scaffolds respectively. These values are very similar to the Young's modulus of living human bone. Chemical treatment did not affect negatively cell proliferation and differentiation. Additionally, the chemically-treated scaffolds had a twofold increase in colonization of osteoblast cells migrating out of multicellular spheroids. Furthermore, X-ray computed microtomography confirmed that chemically-treated scaffolds met the dimensions originally set in the CAD models. Therefore, chemical-treatment can be used as a tool to cancel the discrepancies between the designed and fabricated objects, thus enabling fabrication of finer structures with regular struts and high resolution.

Quantitative stereological analysis of the highly porous hydroxyapatite scaffolds using X-ray CM and SEM.

BACKGROUND: Material properties of the scaffolds as well as their microstructure are vital in determining in vivo cellular response. Three-dimensional (3D), highly porous scaffolds are used in tissue engineering to provide a suitable microenvironment and to support regeneration of bone. Both pore sizes and their architecture, in particular interconnection density, impact functionality of scaffold during its biomedical applications. OBJECTIVE: In this paper a comparative study of the microstructure of highly porous hydroxyapatite scaffolds produced via gelcasting of foamed slurries and replication of polyurethane sponge were carried out. METHODS: Quantitative stereological analysis of the microstructure was conducted using transmission X-ray computed microtomography (µCT) and scanning electron microscopy (SEM). Application of the X-ray microtomography allowed obtaining the 2D cross-sectional images of examined samples, and then the 3D reflection of individual samples. RESULTS: In our studies we proved that the distribution of pores in HAp bioceramics can be controlled by selection of the manufacturing method. In the case of material produced by the gelcasting method, the porosity of the samples was about ∼78 vol.%, while for the method of replication of the porous organic matrix it was higher ∼84 vol.%. Application of gelcasting method resulted in bioceramics with the macropores ranging from 95 µm to 158 µm (the modal value of 120 µm). Furthermore, micropores of size 34 µm-60 µm - so called "windows", were observed on spherical macropores surfaces. In the case of replication of polyurethane sponge only macropores from 295 µm to 337 µm (the modal value of 300 µm) were obtained. Application of µCT and SEM give more information than classical mercury intrusion porosimetry in studies of porous bioceramics. Developed materials met the criteria for porous bone substitutes. CONCLUSIONS: The results of quantitative description of microstructure allowed determining the differences between porous hydroxyapatite bioceramics obtained via replication of porous organic matrix and gelcasting of foamed slurry. The stereological analysis demonstrated, that bioceramics prepared via gelling of foamed slurry has a lower pore size and grains (1.1-1.9 µm) than the material obtained by the method of replication of polyurethane sponge (2.1-2.3 µm). Based on morphological analysis the porosity of tested materials was determined. In the case of material produce by the gelcasting, porosity of the samples was about ∼78 vol.%, while for method of replication of the porous organic matrix the porosity was higher and constituted ∼84 vol.%. Furthermore, evaluated materials varied in porosity and the pore size distribution. It was stated that the method of gelcasting resulted in hydroxyapatite bioceramics with the macropores diameter (95-158 µm), micropores so called "windows" (34-60 µm) - observed on spherical macropores walls and micropores of size 0.6 µm-1.3 µm, which were visible in sintered areas. When the method of replication of polyurethane sponge was applied only macropores from 295 µm to 337 µm were obtained. The comparable values of shape factors such as elongation, curvature of pours boundary and convexity, confirmed that macropores in both studied series had similar shape.

Influencing chondrogenic differentiation of human mesenchymal stromal cells in scaffolds displaying a structural gradient in pore size.

UNLABELLED: Articular cartilage lesions have a limited ability to heal by themselves. Yet, golden standard treatments for cartilage repair such as drilling, microfracture and mosaicplasty provide further damage and an unstable solution that degenerates into fibrocartilage in time. Articular cartilage presents a number of gradients in cell number and size along with structural gradients in extra cellular matrix (ECM) composition. Therefore, creating scaffolds that display a structural gradient can be an appealing strategy for cartilage tissue regeneration treatments. In the present study, a scaffold with an in-built discrete gradient in pore size was produced by additive manufacturing. Human mesenchymal stromal cells (hMSCs) were seeded within the gradient scaffolds and their proliferation, differentiation and ECM deposition was evaluated with respect to 2 non-gradient scaffolds. Glycosaminoglycan (GAG) deposition was significantly higher in gradient scaffolds and non-gradient scaffolds with the smallest pore size compared to non-gradient scaffolds with the largest pore size. A gradual increase of chondrogenic markers was observed within the gradient structures with decreasing pore size, which was also accompanied by an increasingly compact ECM formation. Therefore, scaffolds displaying a structural gradient in pore size seem to be a promising strategy to aid in the process of hMSC chondrogenic differentiation and could be considered for improved cartilage tissue regeneration applications. STATEMENT OF SIGNIFICANCE: We present the development of a novel hierarchical scaffold obtained by additive manufacturing. Structural hierarchy is obtained by changing pore size within the pore network characterizing the fabricated scaffolds and proves to be a functional element in the scaffold to influence adult stem cell differentiation in the chondrogenic lineage. Specifically, in regions of the scaffolds presenting smaller pores an increasing differentiation of stem cells toward the chondrogenic differentiation is displayed. Taking inspiration from the zonal organization of articular cartilage tissue, pore size gradients could, therefore, be considered as a new and important element in designing 3D scaffolds for regenerative medicine applications, in particular for all those tissues where gradient physical properties are present.

Influence of internal pore architecture on biological and mechanical properties of three-dimensional fiber deposited scaffolds for bone regeneration.

Fused deposition modeling has been used to fabricate three-dimensional (3D) scaffolds for tissue engineering applications, because it allows to tailor their pore network. Despite the proven flexibility in doing so, a limited amount of studies have been performed to evaluate whether specific pore shapes have an influence on cell activity and tissue formation. Our study aimed at investigating the influence of internal pore architecture on the biological and mechanical properties of 3D scaffolds seeded with mesenchymal stromal cells. Polycaprolactone scaffolds with six different geometries were fabricated. The 3D samples were manufactured with different lay-down pattern of the fibers by varying the layer deposition angle from 0°/15°/30°, to 0°/30°/60°, 0°/45°/90°, 0°/60°/120°, 0°/75°/150°, and 0°/90°/180°. The scaffolds were investigated by scanning electron microscopy and micro computed tomographical analysis and displayed a fully interconnected pore network. Cell proliferation and differentiation toward the osteogenic lineage were evaluated by DNA, alkaline phosphatase activity, and polymerase chain reaction. The obtained scaffolds had structures with open porosity (50%-60%) and interconnected pores ranging from 380 to 400 µm. Changing the angle deposition affected significantly the mechanical properties of the scaffolds. With increasing the angle deposition between successive layers, the elastic modulus increased as well. Cellular studies also showed influence of the internal architecture on cell adhesion and proliferation within the 3D construct, yet limited influence on cell differentiation was observed.

Post Processing and Biological Evaluation of the Titanium Scaffolds for Bone Tissue Engineering.

Nowadays, post-surgical or post-accidental bone loss can be substituted by custom-made scaffolds fabricated by additive manufacturing (AM) methods from metallic powders. However, the partially melted powder particles must be removed in a post-process chemical treatment. The aim of this study was to investigate the effect of the chemical polishing with various acid baths on novel scaffolds' morphology, porosity and mechanical properties. In the first stage, Magics software (Materialise NV, Leuven, Belgium) was used to design a porous scaffolds with pore size equal to (A) 200 µm, (B) 500 µm and (C) 200 + 500 µm, and diamond cell structure. The scaffolds were fabricated from commercially pure titanium powder (CP Ti) using a SLM50 3D printing machine (Realizer GmbH, Borchen, Germany). The selective laser melting (SLM) process was optimized and the laser beam energy density in range of 91-151 J/mm³ was applied to receive 3D structures with fully dense struts. To remove not fully melted titanium particles the scaffolds were chemically polished using various HF and HF-HNO3 acid solutions. Based on scaffolds mass loss and scanning electron (SEM) observations, baths which provided most uniform surface cleaning were proposed for each porosity. The pore and strut size after chemical treatments was calculated based on the micro-computed tomography (µ-CT) and SEM images. The mechanical tests showed that the treated scaffolds had Young's modulus close to that of compact bone. Additionally, the effect of pore size of chemically polished scaffolds on cell retention, proliferation and differentiation was studied using human mesenchymal stem cells. Small pores yielded higher cell retention within the scaffolds, which then affected their growth. This shows that in vitro cell performance can be controlled to certain extent by varying pore sizes.